JP2008020498A - Automatic focus detection device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an autofocus device utilizable for a substantially transparent non-stained cell or the like. <P>SOLUTION: An automatic focusing device for an imaging apparatus for photographing a subject having light transmittance and refractive index difference is equipped with: a differential image acquiring means for acquiring a differential image from images at a plurality of focal positions; a contrast calculation means for calculating the contrast value of the differential image; and a focusing position decision means for deciding the focal position to which the maximum value of the calculated contrast value is given. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an autofocus device for a substantially transparent subject, and more particularly to an autofocus device for a microscope.

  2. Description of the Related Art Conventionally, in an image observation apparatus such as a microscope, an apparatus that performs an autofocus operation by performing image processing on an observation image captured by an image sensor such as a CCD and determining contrast, a so-called contrast AF type autofocus apparatus is known. It has been.

  In such an apparatus, in the image area to be focused, for example, a contrast value defined by the sum of squares of adjacent pixels can be calculated, and each contrast value is evaluated by changing the focal position. Then, the position where the contrast value is maximized is determined as the in-focus position.

For example, Patent Document 1 describes a contrast AF type focus adjustment device that calculates a contrast value using all or some of the captured pixels.
JP 2002-162558 A

  However, in the observation of phase objects such as unstained cells, there is a problem that the autofocus method as described above cannot be applied because the contrast value of the image at the in-focus position is rather minimal (or minimal). Occur. Due to this problem, there has been no autofocus method that can be applied to subjects such as unstained cells.

  On the other hand, there is a great demand for observing live cells without staining them. One observation technique for this purpose is fluorescence observation. Fluorescence observation is much less damaging to cells than normal staining. However, if the fluorescent material continues to be exposed to excitation light, it will cause a fading phenomenon. Therefore, there is a demand for using the excitation light only in the actual observation stage without using the excitation light in the focusing stage.

  The present invention provides an autofocus device that can be used for phase objects such as unstained cells that are substantially transparent.

  The above-mentioned problem of the present invention is a difference image acquisition means for acquiring a difference image from images at focal positions adjacent to each other at an equal interval in an autofocus device of an imaging device that captures a subject having a light transmittance and a refractive index difference. An autofocus device comprising: a contrast calculation unit that calculates a contrast value of the difference image; and an in-focus position determination unit that determines a focus position that gives the maximum value of the calculated contrast value as the in-focus position. Achieved by using.

  The difference image acquisition means includes an image pickup means for picking up an image, a storage means for storing the picked-up image for at least one screen, and a difference means for obtaining a difference between the stored image and a newly picked-up image. It can comprise by providing.

  The difference image acquisition unit is configured to capture a first imaging unit that captures an image at a first focal position, and a second imaging unit that captures an image at a second focal position that is deviated from the first focal position by a predetermined interval. It can also be configured by including an imaging unit and a difference unit that generates a difference image by taking a difference between an image captured by the first imaging unit and an image captured by the second imaging unit.

  Further, the difference image acquisition means can be configured by acquiring a difference image by guiding the first optical path and the second optical path having a certain optical path difference to a common imaging means.

The imaging means is preferably a CCD (Charge Coupled Devices), and may be a line sensor.
It can be considered that the in-focus position determining means obtains the maximum value of the contrast value by a hill-climbing method. The focal position from which the difference image is acquired may be calculated during focusing control.

  The autofocus device is preferably mounted on an optical microscope. It can also be mounted on live cell observation devices and precision measurement devices.

  According to the present invention, the autofocus device that has the minimum contrast value at the in-focus position and the maximum contrast value of the difference image, and thus functions correctly even in an imaging device that uses a phase object such as an unstained cell as a subject. Is provided.

  Further, as an application thereof, when observing fluorescence of a fluorescently stained subject, it is not necessary to irradiate the excitation light at the stage of focusing, so that the fading phenomenon can be suppressed as much as possible.

Embodiments of the present invention will be described below with reference to the drawings.
FIG. 1 is an overall configuration diagram of the present invention. The microscope, which is one of the embodiments according to the present invention, can be implemented by a general optical microscope, and only the portion directly related to the present invention is shown in the figure. That is, the microscope used for carrying out the present invention may include parts such as a lens, a prism, and an eyepiece that are not shown in the drawing.

  In the example of the microscope shown in FIG. 1, the sample (subject) 2 placed on the stage 3 is illuminated by the light source 1, and the observation light is guided to the image acquisition unit 5 through the objective lens 4. The observation light that has reached the image acquisition unit 5 is converted into an electric signal by an imaging device such as a CCD.

  As will be described later, in the embodiment of the present invention, the image acquisition unit 5 acquires a difference image of images at different focal points, but the image acquisition unit 5 may acquire a normal image. In that configuration, a method of using the acquired image for acquiring observation data of the sample or displaying the screen is conceivable.

  The electrical signal of the difference image obtained by the image acquisition unit 5 is transferred to the image processing unit 6 and a contrast value is calculated. The calculated contrast value is held in a memory or the like in the image processing unit (not shown), and the maximum value is calculated by a method represented by FIG.

  The control unit 7 controls the focal position based on the contrast value calculated by the image processing unit 6. At this time, a method of controlling the stage 3 and a method of controlling the objective lens 4 are conceivable, but either method may be adopted in the present invention.

  FIG. 2 is a schematic diagram showing how unstained cells appear before and after the in-focus position. In the same figure, (c) shows how the unstained cells appear at the in-focus position, (b) shows the appearance before the in-focus position (front pin image), and (a) shows the appearance further in front. Shows the direction. Similarly, (d) and (e) are views (rear pin image) behind the in-focus position.

  As shown in the figure, in the unstained cells, the contrast value is rather low in the observation at the in-focus position, and the contrast value becomes maximum before and after that. That is, a graph of the focal position and the contrast value is shown in FIG.

  Another phenomenon that can be understood from FIG. 2 is that the light and dark positions are reversed in the front pin image (b) and the rear pin image (d). That is, light and dark are amplified in the difference image obtained by taking the difference between the front pin image (b) and the rear pin image (d), and light and dark are canceled out in the difference image between the front pin image (a) and the front pin image (b). A graph of this phenomenon is shown in FIG. In the same figure, the contrast value of the difference image in the focus position of equal intervals is graphed. As can be seen from this figure, the mere contrast value is minimal, but the contrast value of the difference image is maximum at the in-focus position.

  The reason for this phenomenon will be briefly described as follows. As disclosed in Japanese Patent Application Laid-Open No. 2005-218379, there is no phase difference between the light beam diffracted on the cell surface (or the light beam scattered) and the light beam traveling straight, and there is no phase difference at the in-focus position. However, when an optical path difference is produced by shifting the focal position, interference occurs in these light beams. On the other hand, unstained cells are substantially transparent, but have a refractive index difference inside. Thus, the light beam that has passed through the cell has a phase distribution according to the portion that has passed through. As a result, the phase difference due to the shift of the focal position produces light and dark correlated with the phase distribution of the unstained cells, and the unstained cells are visualized. Further, the phase difference caused by shifting the focal position is reversed before and after the focal position, as can be seen from the fact that the phase difference becomes 0 at the focal position. If this is explained in terms of contrast, it means that the contrast of unstained cells is reversed before and after the focal point position.

  In the present invention, by utilizing this phenomenon, an autofocus function capable of determining a focus position even in a substantially transparent subject such as an unstained cell is realized. Note that the above phenomenon applies to all subjects that generate a phase difference. Therefore, the applicable range of the present invention is not limited to unstained cells. For example, it can be used for a measuring instrument for observing fine irregularities formed on a semiconductor substrate, a metal surface or a glass substrate.

  FIG. 5 is a diagram illustrating a first embodiment of the image acquisition unit 5 in the embodiment of the present invention. The incident observation light is converted into electrical information by the image sensor 8 such as a CCD. The image information is temporarily stored in the subsequent image buffer 9. Thereafter, by controlling the observation stage 3 or the objective lens 4, image information at focal positions adjacent to each other at equal intervals is acquired. The image information at different focal points obtained in this way is passed through the image difference processing unit 10 to obtain a difference image. By repeating this process, a difference image is acquired. An advantage of this configuration is that the number of CCDs, which are relatively expensive devices, can be limited to one. In addition, it is possible to easily extract the output from the CCD to another observation apparatus without passing through the difference image processing unit.

  FIG. 6 is a diagram illustrating a second embodiment of the image acquisition unit 5 in the embodiment of the present invention. The incident observation light is separated into two optical paths by the beam splitter 11. Imaging elements 8 such as CCDs are provided at different focal positions each having a certain optical path difference, and image information at the two focal positions is acquired simultaneously. In the subsequent stage, the two image information is passed through the difference image processing unit to obtain a difference image. The advantage of this configuration is that it is possible to obtain a difference image by acquiring images at focal positions adjacent to each other at equal intervals without moving the stage.

  FIG. 7 is a diagram illustrating a third embodiment of the image acquisition unit 5 in the embodiment of the present invention. The incident observation light is separated into two optical paths by the beam splitter 11. After that, the two separated optical paths are given an optical path difference by a lens, a prism, a mirror, or the like, and input to the same image sensor 8. At this time, the two optical paths are set to use half screens of the image sensor 8. Then, the difference image processing unit 10 in the subsequent stage generates a difference image from the two images. The advantage of this configuration is that a difference image can be acquired by a single movement of the focal position while the number of CCDs, which are relatively expensive devices, can be limited to one. It is also expected to be suitable for accurate measurement by eliminating individual differences such as CCD.

FIG. 8 illustrates a method for obtaining the maximum contrast value of the difference image in the embodiment of the present invention. This method uses a so-called hill-climbing method.
First, a sensor such as a CCD is read to acquire images with different focal positions (S1). Next, difference data (difference image) for each pixel is created from the acquired image (S2). Thereafter, the contrast value of the difference image is calculated (S3), and the result is stored in the memory (S4).

  As the next step, the focus position is moved by controlling the stage and the objective lens (S5). Similarly, an image is acquired, difference data is created, and a contrast value is calculated (S6-S8). Thereafter, the contrast value stored in the memory is compared, and if it is larger than this value (S9 is No), the process returns to S4 to update the stored contrast value.

  If the contrast value is smaller than that stored in the memory (Yes in S9), it is understood that the focus position one step before was the maximum contrast value. Therefore, the focal point can be found by returning the focal position by one step.

  Note that the method of determining the maximum contrast value in the present invention is not limited to the above-described hill-climbing method. For example, after executing the above-described hill-climbing method, a method of increasing the accuracy of the in-focus position by measuring the contrast value by narrowing the interval between the focus positions from which images are acquired can be considered. A method of shortening the focusing time is also conceivable if the focal position is predicted from the measured change in contrast value and the interval between the focal positions at which images are acquired is controlled. Further, after all images at the focal positions at equal intervals are first acquired, respective difference images may be generated to obtain the maximum of these contrast values. This method is expected to reliably determine the maximum point.

  Finally, a supplementary explanation will be given regarding the method of calculating the contrast value. There are various definitions of numerical values called contrast values. In a simple example, the contrast value is determined by the difference between the brightness of the brightest part and the brightness of the darkest part. That is, the contrast value C is given by the following equation.

Here, I _j represents the luminance for each pixel. In general, the definition of the contrast includes an absolute value and a maximum value. As a result, the order of S2 and S3 (or S7 and S8) in the flowchart of FIG. 8 cannot be changed. That is, the “contrast value of the difference image between the two images” and the “difference between the contrast values of the two images” are different.

  With the above configuration, an autofocus device is also provided in an imaging apparatus that uses a substantially transparent object such as an unstained cell as a subject.

It is a whole block diagram of the Example of this invention. It is a figure which shows how the unstained cell looks with the front pin, a focus position, and a back pin. It is a graph explaining the relationship between a focus position and a contrast value. It is a graph explaining the relationship between a focus position and the contrast value of a difference image. It is a figure explaining 1st embodiment of an image acquisition part. It is a figure explaining 2nd embodiment of an image acquisition part. It is a figure explaining 3rd embodiment of an image acquisition part. It is a figure explaining the mountain climbing method in the Example of this invention.

Explanation of symbols

1 ... light source 2 ... sample (subject)
DESCRIPTION OF SYMBOLS 3 ... Stage 4 ... Objective lens 5 ... Image acquisition part 6 ... Image processing part 7 ... Control part 8 ... Imaging element 9 ... Image buffer 10 ... Difference image processing Part 11: Beam splitter 12: Lens / prism

Claims (12)

In an autofocus device of an imaging device that captures a subject having a light transmittance and a refractive index difference,
Differential image acquisition means for acquiring a differential image from images at focal positions adjacent at equal intervals;
Contrast calculation means for calculating a contrast value of the difference image;
An automatic focusing apparatus, comprising: an in-focus position determining unit that determines a focus position that gives the calculated maximum contrast value as an in-focus position. The difference image acquisition means includes
An imaging means for capturing an image;
Storage means for storing the captured image for at least one screen;
2. The autofocus device according to claim 1, further comprising difference means for obtaining a difference between the stored image and a newly captured image. The difference image acquisition means includes
First imaging means for imaging an image at a first focal position;
Second imaging means for imaging an image at a second focal position that is deviated from the first focal position by a fixed interval;
The autofocus device according to claim 1, further comprising a difference unit that generates a difference image by taking a difference between an image captured by the first image capturing unit and an image captured by the second image capturing unit. . The difference image acquisition means includes
2. The autofocus device according to claim 1, wherein the difference image is obtained by guiding the first optical path and the second optical path having a constant optical path difference to a common imaging unit.   5. The autofocus device according to claim 2, wherein the imaging means is a CCD (Charge Coupled Devices).   5. The autofocus device according to claim 2, wherein the image pickup unit is a line sensor.   7. The automatic focusing apparatus according to claim 1, wherein the in-focus position determining unit obtains the maximum value of the contrast value by a hill-climbing method.   The autofocus device according to any one of claims 1 to 6, wherein a focus position at which the difference image is acquired is calculated during focus control.   An optical microscope comprising the autofocus device according to any one of claims 1 to 8.   A live cell observation device comprising the autofocus device according to any one of claims 1 to 8.   A precision measuring device comprising the autofocus device according to any one of claims 1 to 8. In a method for determining a focal point position of an imaging device that has a light transmission property and captures an object having a refractive index difference therein,
Take images at multiple focal positions,
A difference image is generated from two images at adjacent focal positions among the images,
Calculate the contrast of the generated difference image,
By determining the difference image that maximizes the calculated contrast,
A method for determining the in-focus position.